Epigenetic control of vascular niche capacity to support hematopoiesis.

项目总结/摘要 造血是一种细胞发育过程，受细胞内在发育程序和来自微环境的细胞外在因素控制。存在于骨髓和其他造血器官中的血管龛提供了适当调节该过程所必需的物理空间和信号。接受化疗或造血干细胞移植（HSCT）的患者的造血干细胞（HSC）系统发育和功能多样性受到限制，导致克隆性造血和血细胞减少，这是这些患者发病率和死亡率的重要来源。目前，对微环境在调节HSC多样性中的作用知之甚少。我们假设，机制存在于血管生态位内，可以调节，使其支持一个更具遗传学和功能多样性的HSC群体。我们在条形码GESTALT斑马鱼中进行了遗传筛选，发现prkcda（斑马鱼PKC-δ同源物）的表达失调使造血干细胞克隆的数量增加了50%以上。使用单细胞RNA-seq的表型分析证明存在新的未成熟中性粒细胞群体和红系前体细胞的扩增。单细胞ATAC-seq分析表明，天然prkcda基因座启动子处的染色质可及性在血管小生境细胞群体中特异性降低。这些数据使我们假设存在一个特定的表观遗传程序内的血管生态位，调节支持造血的因子的表达，从而保持生态位的能力，以支持HSC的系统发育和功能多样性。在目标1中，我们将使用特定表观遗传因子的"切和运行"分析来表征人内皮细胞培养物中的血管生态位。多模式单细胞RNA-seq/ATAC-seq将用于成年和胚胎斑马鱼，以鉴定小生境细胞群和支持造血的重要基因附近的顺式作用DNA元件。在Aim 2中，CRISPR筛选将用于鉴定人类血管龛细胞中的特异性转录因子结合位点，该位点邻近并控制参与龛功能的关键基因的转录，包括CXCL12，ANG1和KITLG。将制作一组斑马鱼CRISPR突变体，以研究所选增强子元件在体内的功能。将通过与HSPC共培养在体外和通过GESTALT条形码和scRNA-seq在体内测定生态位功能的变化。这项工作将提供新的机制理解造血生态位的能力，以支持不同的造血干细胞群体是如何调节，并可能导致新的疗法，改善化疗或移植后的造血恢复。

PROJECT SUMMARY/ABSTRACT Hematopoiesis is a cellular developmental process that is controlled by a cell-intrinsic developmental program and cell-extrinsic factors from the microenvironment. The vascular niche present in the bone marrow and other hematopoietic organs provides the physical space and signals necessary for the proper regulation of this process. Hematopoietic stem cell (HSC) phylogenetic and functional diversity are restricted in patients who receive chemotherapy or are recipients of hematopoietic stem cell transplantation (HSCT), leading to clonal hematopoiesis and cytopenias which are significant sources of morbidity and mortality in these patients. At present, very little is known about the role of the microenvironment in regulating HSC diversity. We hypothesized that mechanisms exist within the vascular niche that can be modulated to allow it to support a more phylogenetically and functionally diverse population of HSCs. We performed a genetic screen in barcoded GESTALT zebrafish and found that dysregulated expression of prkcda, the zebrafish PKC-delta homolog, increased the number of HSC clones contributing to hematopoiesis by more than 50%. Phenotypic analysis using single cell RNA-seq demonstrated the presence of a novel population of immature neutrophils and expansion of erythroid precursor cells. Single cell ATAC-seq analysis showed that chromatin accessibility at the promoter of the native prkcda locus is reduced specifically in vascular niche cell populations. These data led us to hypothesize the existence of a specific epigenetic program within the vascular niche that regulates expression of factors supporting hematopoiesis and thereby maintains the capacity of the niche to support HSC phylogenetic and functional diversity. In Aim 1, we will use Cut and Run analysis for specific epigenetic factors to characterize the vascular niche in human endothelial cell cultures. Multimodal single cell RNA-seq/ATAC-seq will be used in adult and embryonic zebrafish to identify niche cell populations and cis-acting DNA elements near genes important for supporting hematopoiesis. In Aim 2, a CRISPR screen will be used to identify specific transcription factor binding sites in human vascular niche cells lying proximal to and controlling transcription of key genes involved in niche function, including CXCL12, ANG1, and KITLG. A panel of zebrafish CRISPR mutants will be made to study the function of selected enhancer elements in vivo. Changes in niche function will be assayed in vitro by co-culture with HSPCs and in vivo by GESTALT barcoding and scRNA-seq. This work will provide new mechanistic understanding of how the capacity of the hematopoietic niche to support a diverse population of HSCs is regulated and may lead to new therapies that improve hematopoietic recovery after chemotherapy or transplantation.